C. jejuni is a leading cause of diarrheal disease worldwide and a documented threat to US military personnel (Taylor, Current status and future trends. Amer. Soc. Micro., (1992); Tauxe, Current status and future trends. Amer. Soc. Micro. 1992). The symptoms of campylobacter enteritis include diarrhea, abdominal pain, and fever and often accompanied by vomiting. Stools usually contain mucus, fecal leukocytes, and blood, although watery diarrhea is also observed (Cover and Blaser, Ann. Rev. Med., 40: 269-285 (1999). However, despite the importance of this organism to human disease, there are no licensed vaccines against C. jejuni. 
Because of the medical importance of C. jejuni, considerable research is dedicated toward understanding the pathogen. However, notwithstanding this effort, there is surprisingly little understanding about how C. jejuni causes human disease. The genome of one strain, NCTC 11168 (Parkhill, et al., Nature, 403: 665-668 (2000) revealed several unusual aspects about the biology of C. jejuni. One striking feature is the presence of an unexpectedly high number of genes encoding putative enzymes involved in sugar and/or polysaccharide synthesis (Parkhill et al., Nature, 403: 665-668 (2000). The sequence, and resulting research fostered primarily by the availability of the sequence, has revealed that these genes fall into 4 main functional clusters that underscore the importance of some unusual carbohydrate structure to the biology of C. jejuni. These clusters include Lipooligosaccharide (LOS) synthesis, genetic control of flagellin glycosylation, genetic control of N-linked glycosylation, and the control of the biosynthesis and assembly of capsule.
Vaccine strategies against C. jejuni have been largely limited due to the molecular mimicry between lipooligosaccharide (LOS) cores of many strains of C. jejuni and human gangliosides (Moran, et al., J. Endotox. Res., 3: 521-531 (1996). This mimicry is thought to be a major factor in the strong association of C. jejuni infection with Guillain Barre Syndrome (GBS), a post-infectious polyneuropathy (Allos, J. Infect. Dis., 176(Suppl.): S125-128 (1997)). Thus, antibodies generated against LOS cores result in an autoimmune response to human neural tissue. It has been estimated that as many as 1/3000 cases of campylobacter enteritis results in GBS. Therefore, the possibility of developing GBS could be associated with any whole cell vaccine against C. jejuni that includes ganglioside mimicry.
LOS synthesis in Campylobacter is controlled by a number of genes, including genes encoding enzymes involved in biosynthesis of sialic acid for incorporation into LOS. Thus, C. jejuni is one of a limited number of bacteria that can endogenously synthesize sialic acid, a 9 carbon sugar that is found in many mammalian cells. This is consistent with the observed molecular mimicry of LOS and human gangliosides important in GBS (Aspinall, et al., Eur. J. Biochem., 213: 1029-1037 (1993); Aspinall, et al., Infect. Immun. 62: 2122-2125 (1994); Aspinall, et al., Biochem., 33: 241-249 (1994); Salloway et al., Infect. Immun., 64: 2945-2949 (1996)).
Although glycosylation of proteins was once considered to be a eukaryotic trait, there is an increase awareness of prokaryotic protein glycosylation (Power and Jennings, FEMS Microbiol. Lett., 218: 211-222 (2003); Ewing, et al., J. Bacteriol. 191:7086 (2009)). The best characterized and most extensively glycosylated bacterial protein is campylobacter flagellin. Flagellin from strain 81-176 is glycosylated at 19 serine or threonine sites by an O-linkage to pseudaminic acid and derivatives of pseudaminic acid (Thibault et al., J. Biol. Chem., 276: 34862-34870 (2001)). Pseudaminic acid is an unusual 9 carbon sugar that resembles sialic acid, but which is highly immunogenic, unlike sialic acid. Moreover, mutants that are unable to glycosylate flagellin cannot assemble a flagellar filament (Goon et al, Mol. Microbiol., 50: 659-671 (2003)). Since flagella are indispensable virulence determinants of C. jejuni, glycosylation is therefore also a key virulence determinant.
One of the most unusual aspects of C. jejuni is the presence of a general system for N-linked glycosylation of numerous proteins (Szymanski et al., Mol. Microbiol., 32: 1022-1030 (1999)); reviewed in Szymanski et al., Trends Microbiol., 11: 233-238 (2003). This system, which includes an oligosaccharide transferase similar to that found in the eukaryote Saccharomyces cerevisiae, attaches a glycan which has recently been shown to be a heptasaccharide composed of one bacillosamine residue (an unusual deoxy sugar), one D-glucose, and five D-GalNAc residues (Young et al., J. Biol. Chem., 277: 42530-42539 (2002)). The glycosylation appears to occur on numerous periplasmic, and perhaps, surface exposed proteins in C. jejuni (Young et al., J. Biol. Chem., 277: 42530-42539 (2002)). The unusual glycan, again, appears to be highly immunogenic and is recognized during human infection (Szymanski et al., Mol. Microbiol., 32: 1022-1030 (1999); Szymanski et al., Trends Microbiol., 11: 233-238 (2003)).
An interesting recent revelation regarding the Campylobacter genome sequence was the presence of a complete set of capsule transport genes similar to those seen in type II/III capsule loci in the Enterobactericeae (Parkhill et al., Nature, 403: 665-668 (2000); Karlyshev et al., Mol. Microbiol., 35: 529-541 (2000)). Subsequent genetic studies in which site-specific mutations were made in several capsule transport genes indicated that the capsule was the serodeterminant of the Penner serotyping scheme (Karlyshev et al., Mol. Microbiol., 35: 529-541 (2000)). The Penner scheme (or HS for heat stable) is one of two major serotyping schemes of campylobacters and was originally thought to be based on lipopolysaccharide O side chains (Moran and Penner, J. Appl. Microbiol., 86: 361-377 (1999)).
Currently it is believed that the structures previously described as O side chains are, in fact, capsules. The chemical structures of the capsule/O side chains of several Penner serotypes have been determined, and these structures include several unusual sugar structures, as summarized in Table 1.
TABLE 1Structure of some capsular polysaccharides of C.jejuni strains.StrainStructureReferenceHS3→4-α-D-Gal-(1→3)(3-hydroxypropanoyl)-L-glycero-α-D-ido-Hep-(1→Aspinall et al.,1995HS19→4)-β-D-GlcA-(1→3)-β-D-GlcNAc-(1→Aspinall et al.,(the GlcA units are present as amides of 2-amino-2-deoxyglycerol)1994 a, bHS23, HS36Four closely-related polysaccharides:Aspinall et al.,→3)-β-D-GlcNAc-(1→3)-α-D-Gal-(1→2)-6d-α-D-altro-Hep-(1→;1992→3)-β-D-GlcNAc-(1→3)-α-D-Gal-(1→2)-6d-3-O—Me-α-D-altro-Hep-(1→;→3)-β-D-GlcNAc-(1→3)-α-D-Gal-(1→2)-D-glycero-α-D-altro-Hep-(1→;→3)-β-D-GlcNAc-(1→3)-α-D-Gal-(1→2)-3-O—Me-D-glycero-α-D-altro-Hep-(1→ 81116Muldoon et al. (2002) NCTC11168St. Michael et al. (2002)
There currently are no licensed vaccines against Campylobacter, due greatly to the molecular mimicry between LOS cores of many strains of C. jejuni and human gangliosides (Moran, et al., J. Endotox. Res., 3: 521-531 (1996)). However, vaccine formulations incorporating bacterial capsules have been developed against a number of pathogens. In general, capsule vaccines are immunogenic in humans and non-toxic (Jennings, Curren. Top. Microbiol. Immunol., 150: 97-127 (1990)). One of the general problems associated with capsule vaccines is the poor immunogenicity of all polysaccharides in infants, and the fact that many of the capsular vaccines are directed at diseases that are particular threatening to the pediatric population. Based on murine studies, pure polysaccharide antigens are considered to be T cell independent, and capable of inducing only IgM type responses. Adult humans, in contrast, are able to generate IgG, in addition to IgM and IgA antibodies against polysaccharides. Responses in infants to vaccines against type B H. influenzae (Schneerson et al., J. Exp. Med. 152: 361-376 (1980); Anderson, P. W., Infect. Immun., 39: 233-238 (1983); Marburg, et al., J. Am. Chem. Soc., 108: 5282-5287 (1986)), group A, B and C Neisseria meningiditis (Jennings and Lugowski, J. Immunol., 127: 1011-1018 (1981); and Jennings, et al., Carbohydr. Res., 112: 105-111 (1983)); and type 6A Streptococcus pneumoniae (Chu et al., Infect. Immun. 40: 245-256 (1983)) have all improved following conjugation to proteins.
C. jejuni capsule, as defined in this application, is a generic term for capsular polymers, which are composed of repeating polysaccharide structures. The repeating structures can be homopolymers, defined as a repeating single sugar moiety, or repeating oligosaccharides (i.e. disaccharides or trisaccharides, etc.). A number of species of capsular repeating polysaccharide polymers have been identified. To illustrate the genus of capsular polysaccharide structures, Table 2 lists known capsular polysaccharide structures for Campylobacter strains.
TABLE 2Strain/HStypeStructureReferenceHS3→4-α-D-Gal-(1→3)(3-hydroxypropanoyl)-L-glyero-α-D-ido-Hep-(1→Aspinall, etal (1995HS19→4)-β-D-GlcA-(1→)-β-D-GlcNAc-(1→Aspinall, et(GlcA units are present as amides of 2-amino-2-deoxyglycerol)al., 1994 (aand b)HS23/36Four closely-related polysaccharides:Aspinall, et→3)-β-D-GlcNAc-(1→3)-α-D-Gal-(1→2)-6d-α-D-altro-Hep-(1→;al., 1992→3)-β-D-GlcNAc-(1→3)-α-D-Gal-(1→2)-6d-3-O-Me-α-D-altro-Hep-(1→;→3)-β-D-GIcNAc-(1→3)-α-D-Gal-(1→2)-D-glycero-α-D-altro-Hep-(1→;→3)-β-D-GlcNAc-(1→3)-α-D-Gal-(1→2)-3-O-Me-D-glycero-α-D-altro-Hep(1→ 81116Muldoon, et al., 2002 NCTC 11168(HS2)St. Michael, et al., 2002 HS41Two closely related polysaccharides:→2)-β-L-Araf-(1→2)-β-D-6d-altro-Hepf-(1→2)-β-L-6d-altrof-(1→(75%; andHannify, et→2)-β-L-Araf-(1→2)-β-D-6d-altro-Hepf-(1→2)-α-D-Fucf-(1→(25%)al., 1999 HS30 (C. coli)Aspinall, et al., 1993 HS1→4)-β-D-Gal-(1→2)-(R)-Gro-(1-P→Aspinall(with two branches at C-2 and C-3 of Gal of β-D fructofuranoses that are further1998;substituted at C-3 with O-methyl phosphoramidate groupsMcNally, etal., 2005HS 4, 13,→3)-6-d-β-D-ido-Hep-(1→4)-β-D-GlcNAc-(1→Chen, et al.64With O-methyl phosphoramidate units present in non-stoichiometric amounts at the2008(GC8486)O-2 and/or O-7 positions of 6-deoxy-beta-D-ido-Heptose. 81-176 (HS23/36)Kanipes, et al., 2006